Resistance training-induced muscle anabolism and subsequent hypertrophy occur most rapidly during the early phase of training and become progressively slower over time. Currently, little is known about the intracellular signaling mechanisms underlying changes in the sensitivity of muscles to training stimuli. We investigated the changes in the exercise-induced phosphorylation of hypertrophic signaling proteins during chronic resistance training and subsequent detraining. Male rats were divided into 4 groups: 1 bout (1B), 12 bouts (12B), 18 bouts (18B), and detraining (DT). In the DT group, rats were subjected to 12 exercise sessions, detrained for 12 days, and then were subjected to 1 exercise session before being sacrificed. Isometric training consisted of maximum isometric contraction was produced by percutaneous electrical stimulation of the gastrocnemius muscle every other day. Muscles were removed 24 h after the final exercise session. Levels of total and phosphorylated p70S6K, 4E-BP1, rpS6, and p90RSK levels were measured, and phosphorylation of p70S6K, rpS6, and p90RSK was elevated in the 1B group compared to control muscle (CON) after acute resistance exercise, while repeated bouts of exercise suppressed those phosphorylation in both 12B and 18B groups. Interestingly, these phosphorylation levels were restored following 12 days of detraining in the DT group. On the contrary, phosphorylation of 4E-BP1 was not altered with chronic training and detraining, indicating that with chronic resistance training, anabolic signaling becomes less sensitive to resistance exercise stimuli, but is restored after a short detraining period.